The neuronal Na+-dependent glutamate transporter, excitatory amino acid carrier 1 (EAAC1, also called EAAT3), has been implicated in the control of synaptic spillover of glutamate, synaptic plasticity, and the import of cysteine for neuronal synthesis of glutathione. EAAC1 protein is observed in both perisynaptic regions of the synapse and in neuronal cell bodies. Although amino acid residues in the carboxyl terminal tail have been implicated in the dendritic targeting of EAAC1 protein, it is not known if mRNA for EAAC1 may also be targeted to dendrites. Sorting of mRNA to specific cellular domains provides a mechanism by which signals can rapidly increase translation in a local environment; this form of regulated translation has been linked to diverse biological phenomena ranging from establishment of polarity during embryogenesis to synapse development and synaptic plasticity. In the present study, EAAC1 mRNA sequences were amplified from dendritic samples that were mechanically harvested from low-density hippocampal neuronal cultures. In parallel analyses, mRNA for histone deacetylase 2 (HDAC-2) and glial fibrillary acidic protein (GFAP) was not detected, suggesting that these samples are not contaminated with cell body or glial mRNAs. EAAC1 mRNA also co-localized with Map2a (a marker of dendrites) but not Tau1 (a marker of axons) in hippocampal neuronal cultures by in situ hybridization. In control rats, EAAC1 mRNA was observed in soma and proximal dendrites of hippocampal pyramidal neurons. Following pilocarpine- or kainate-induced seizures, EAAC1 mRNA was present in CA1 pyramidal cell dendrites up to 200 μm from the soma. These studies provide the first evidence that EAAC1 mRNA localizes to dendrites and suggest that dendritic targeting of EAAC1 mRNA is increased by seizure activity and may be regulated by neuronal activity/depolarization.
glutamate transport; EAAC1; EAAT3; epilepsy; pilocarpine; seizure; mRNA targeting
Glutathione (GSH) is a tripeptide consisting of glutamate, cysteine, and glycine; it has a variety of functions in the central nervous system. Brain GSH depletion is considered a preclinical sign in age-related neurodegenerative diseases, and it promotes the subsequent processes toward neurotoxicity. A neuroprotective mechanism accomplished by increasing GSH synthesis could be a promising approach in the treatment of neurodegenerative diseases. In neurons, cysteine is the rate-limiting substrate for GSH synthesis. Excitatory amino acid carrier 1 (EAAC1) is a neuronal cysteine/glutamate transporter in the brain. EAAC1 translocation to the plasma membrane promotes cysteine uptake, leading to GSH synthesis, while being negatively regulated by glutamate transport associated protein 3-18 (GTRAP3-18). Our recent studies have suggested GTRAP3-18 as an inhibitory factor for neuronal GSH synthesis. Inhibiting GTRAP3-18 function is an endogenous mechanism to increase neuron-specific GSH synthesis in the brain. This review gives an overview of EAAC1-mediated GSH synthesis, and its regulatory mechanisms by GTRAP3-18 in the brain, and a potential approach against neurodegeneration.
glutathione; cysteine uptake; GTRAP3-18; EAAC1; neurodegeneration
Excitatory amino acid carrier 1 (EAAC1) is a glutamate transporter found in neuronal tissues and is extensively expressed in the retina. EAAC1 plays a role in a variety of neural functions, but its biological functions in the retina has not been fully determined. The purpose of this study was to identify proteins regulated by EAAC1 in the retina of mice. To accomplish this, we used a proteomics-based approach to identify proteins that are up- or down-regulated in EAAC1-deficient (EAAC1-/-) mice.
Proteomic analyses and two-dimensional gel electorphoresis were performed on the retina of EAAC1-/- mice, and the results were compared to that of wild type mice. The protein spots showing significant differences were selected for identification by mass spectrometric analyses. Thirteen proteins were differentially expressed; nine proteins were up-regulated and five proteins were down-regulated in EAAC1-/- retina. Functional clustering showed that identified proteins are involved in various cellular process, e.g. cell cycle, cell death, transport and metabolism.
We identified thirteen proteins whose expression is changed in EAAC-/- mice retinas. These proteins are known to regulate cell proliferation, death, transport, metabolism, cell organization and extracellular matrix.
The predominant neuronal glutamate transporter, EAAC1 (for excitatory amino acid carrier-1), is localized to the dendrites and somata of many neurons. Rare presynaptic localization is restricted to GABA terminals. Because glutamate is a precursor for GABA synthesis, we hypothesized that EAAC1 may play a role in regulating GABA synthesis and, thus, could cause epilepsy in rats when inactivated. Reduced expression of EAAC1 by antisense treatment led to behavioral abnormalities, including staring–freezing episodes and electrographic (EEG) seizures. Extracellular hippocampal and thalamocortical slice recordings showed excessive excitability in antisense-treated rats. Patch-clamp recordings of miniature IPSCs (mIPSCs) conducted in CA1 pyramidal neurons in slices from EAAC1 antisense-treated animals demonstrated a significant decrease in mIPSC amplitude, indicating decreased tonic inhibition. There was a 50% loss of hippocampal GABA levels associated with knockdown of EAAC1, and newly synthesized GABA from extracellular glutamate was significantly impaired by reduction of EAAC1 expression. EAAC1 may participate in normal GABA neurosynthesis and limbic hyperexcitability, whereas epilepsy can result from a disruption of the interaction between EAAC1 and GABA metabolism.
EAAC1; transport; antisense; GABA; metabolism; epilepsy
Recently, we demonstrated that mRNA for the neuronal glutamate transporter, excitatory amino acid carrier 1 (EAAC1), is found in dendrites of hippocampal neurons in culture and in dendrites of hippocampal pyramidal cells after pilocarpine-induced status epilepticus (SE). We also showed that SE increased the levels of EAAC1 mRNA ~15-fold in synaptoneurosomes. In the present study, the effects of SE on the distribution EAAC1 protein in hippocampus were examined. In addition, the effects of Group 1 mGluR receptor activation on the levels of EAAC1 protein were examined in synaptoneurosomes prepared from sham control animals and from animals that experience pilocarpine-induced SE. We find that EAAC1 immunoreactivity increases in pyramidal cells of the hippocampus after 3 h of SE. In addition, the group I mGluR agonist, (S)-3,5-dihydroxyphenylglycine (DHPG), caused an increase in EAAC1 protein levels in hippocampal synaptoneurosomes; this effect of DHPG was much larger (~3- to 5-fold) after 3 h of SE. The DHPG-induced increases in EAAC1 protein were blocked by two different inhibitors of translation but not by inhibitors of transcription. mGluR1 or mGluR5 antagonists completely blocked the DHPG-induced increases in EAAC1 protein. DHPG also increased the levels of GluR2/3 protein, but this effect was not altered by SE. The DHPG-induced increase in EAAC1 protein was blocked by an inhibitor of the mammalian target of rapamycin (mTOR) or an inhibitor of extracellular signal-regulated kinase (ERK). These studies provide the first evidence EAAC1 translation can be regulated, and they show that regulated translation of EAAC1 is up-regulated after SE.
glutamate transport; EAAC1; epilepsy; pilocarpine; seizure; mGluR; dendritic targeting
Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na+-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production.
Disturbed glutamate homeostasis may contribute to the pathological processes involved in Alzheimer’s disease (AD). Once glutamate is released from synapses or from other intracellular sources, it is rapidly cleared by glutamate transporters. EAAC1 (also called EAAT3 or SLC1A1) is the primary glutamate transporter in forebrain neurons. In addition to transporting glutamate, EAAC1 plays other roles in regulating GABA synthesis, reducing oxidative stress in neurons, and is important in supporting neuron viability. Currently, little is known about EAAC1 in AD. To address whether EAAC1 is disturbed in AD, immunohistochemistry was performed on tissue from hippocampus and frontal cortex of AD and normal control subjects matched for age and gender. While EAAC1 immunostaining in cortex appeared comparable to controls, in the hippocampus, EAAC1 aberrantly accumulated in the cell bodies and proximal neuritic processes of CA2–CA3 pyramidal neurons in AD patients. Biochemical analyses showed that Triton X-100-insoluble EAAC1 was significantly increased in the hippocampus of AD patients compared to both controls and Parkinson’s disease patients. These findings suggest that aberrant glutamate transporter expression is associated with AD-related neuropathology and that intracellular accumulation of detergent-insoluble EAAC1 is a feature of the complex biochemical lesions in AD that include altered protein solubility.
Glutamate Uptake; Glutamate toxicity; Synaptic dysfunction; protein aggregation; neurodegeneration; memantine; excitotoxicity
Transmembrane glutamate transport by the excitatory amino acid carrier (EAAC1) is coupled to the cotransport of three Na+ ions and one proton. Previously, we suggested that the mechanism of H+ cotransport involves protonation of the conserved glutamate residue E373. However, it was also speculated that the cotransported proton is shared in a H+-binding network, possibly involving the conserved histidine 295 in the 6th transmembrane domain of EAAC1. Here, we used site-directed mutagenesis together with pre-steady state electrophysiological analysis of the mutant transporters in order to test the protonation state of H295 and to determine its involvement in proton transport by EAAC1. Our results show that replacement of H295 with glutamine, an amino acid residue that can not be protonated, generates a fully functional transporter with transport kinetics that are close to the wild-type EAAC1. In contrast, replacement with lysine results in a transporter in which substrate binding and translocation are dramatically inhibited. Furthermore, it is demonstrated that the effect of the histidine 295 to lysine mutation on the glutamate affinity is caused by its positive charge, since wild-type-like affinity can be restored by changing the extracellular pH to 10.0, thus partially deprotonating H295K. Together, these results suggest that histidine 295 is not protonated in EAAC1 at physiological pH, and, thus, does not contribute to H+ cotransport. This conclusion is supported by data from H295C-E373C double mutant transporters which demonstrate that these residues can not be linked by oxidation, indicating that H295 and E373 are not close in space and do not form a proton binding network. A kinetic scheme is used to quantify the results, which includes binding of the cotransported proton to E373 and binding of a modulatory, non-transported proton to the amino acid side chain in position 295.
Glutamate; excitatory amino acid transporter; EAAC1; glutamate transporter; mutagenesis; pre-steady-state kinetics
Glutamate is a regulated molecule in the mammalian testis. Extracellular regulation of glutamate in the body is determined largely by the expression of plasmalemmal glutamate transporters. We have examined by PCR, western blotting and immunocytochemistry the expression of a panel of sodium-dependent plasmalemmal glutamate transporters in the rat testis. Proteins examined included: glutamate aspartate transporter (GLAST), glutamate transporter 1 (GLT1), excitatory amino acid carrier 1 (EAAC1), excitatory amino acid transporter 4 (EAAT4) and EAAT5. We demonstrate that many of the glutamate transporters in the testis are alternately spliced. GLAST is present as exon-3- and exon-9-skipping forms. GLT1 was similarly present as the alternately spliced forms GLT1b and GLT1c, whereas the abundant brain form (GLT1a) was detectable only at the mRNA level. EAAT5 was also strongly expressed, whereas EAAC1 and EAAT4 were absent. These patterns of expression were compared with the patterns of endogenous glutamate localization and with patterns of 𝒹-aspartate accumulation, as assessed by immunocytochemistry. The presence of multiple glutamate transporters in the testis, including unusually spliced forms, suggests that glutamate homeostasis may be critical in this organ. The apparent presence of many of these transporters in the testis and sperm may indicate a need for glutamate transport by such cells.
excitatory amino acid transporter; glutamate aspartate transporter; glutamate transporter 1; sperm; splice variant; testis; transporter
The excitatory amino acid carrier EAAC1 belongs to a family of glutamate transporters that use the electrochemical transmembrane gradients of sodium and potassium to mediate uphill transport of glutamate into the cell. While the sites of cation interaction with EAAC1 are unknown, two cation binding sites were observed in the crystal structure of the bacterial glutamate transporter homologue GltPh. Although occupied by Tl+ in the crystal structure, these sites were proposed to be Na+ binding sites. Therefore, we tested whether Tl+ has the ability to replace Na+ also in the mammalian transporters. Our data demonstrate that Tl+ can bind to EAAC1 with high affinity and mediate a host of different functions. Tl+ can functionally replace potassium when applied to the cytoplasm and support glutamate transport current. When applied extracellularly, Tl+ induces some behavior that mimics that of the Na+-bound transporter, such as activation of the cation-induced anion conductance and creation of a substrate binding site, but it cannot replace Na+ in supporting glutamate transport current. Moreover, our data show a differential effect of mutations to two acidic amino acids potentially involved in cation binding (D367 and D454) on Na+ and Tl+ affinity. Overall, our results demonstrate that the ability of the glutamate transporters to interact with Tl+ is conserved between GltPh and a mammalian member of the transporter family. However, in contrast to GltPh, which does not bind K+, Tl+ is more efficient in mimicking K+ than Na+ when interacting with the mammalian protein.
The excitatory amino acid transporters (EAATs), or sodium-dependent glutamate transporters, provide the primary mechanism for glutamate removal from the synaptic cleft. EAAT distribution has been determined in the rat brain, but it is only partially characterized in the spinal cord.
The regional anatomic distribution of EAATs in spinal cord was assessed by radioligand autoradiography throughout cervical, thoracic, and lumbar cord levels in female Sprague-Dawley rats. EAAT subtype regional distribution was evaluated by inclusion of pharmacologic transport inhibitors in the autoradiography assays and by immunohistochemistry using subtype-specific polyclonal antibodies to rat GLT1 (EAAT2), GLAST (EAAT1), and EAAC1 (EAAT3) rat transporter subtypes.
[3H]-D-Aspartate binding was distributed throughout gray matter at the 3 spinal cord levels, with negligible binding in white matter. Inclusion of pharmacologic transport inhibitors indicates that the EAAT2/GLT1 subtype represents 21% to 40% of binding. Both EAAT1/GLAST and EAAT3/EAAC1 contributed the remainder of binding. Immunoreactivity to subtype-specific antibodies varied, depending on cord level, and was present in both gray and white matter. All 3 subtypes displayed prominent immunoreactivity in the dorsal horn. EAAT3/EAAC1 and to a lesser extent EAAT1/GLAST immunoreactivity also occurred in a punctate pattern in the ventral horn.
The results indicate heterogeneity of EAAT distribution among spinal cord levels and regions. The presence of these transporters throughout rat spinal cord suggests the importance of their contributions to spinal cord function.
Spinal cord; Glutamate plasma membrane proteins; Autoradiography; Immunohistochemistry; Amino acid transporters, Excitatory
RTN1A is a reticulon protein with predominant localization in the endoplasmic reticulum (ER). It was previously shown that RTN1A is expressed in neurons of the mammalian central nervous system but functional information remains sparse. To elucidate the neuronal function of RTN1A, we chose to focus our investigation on identifying possible novel binding partners specifically interacting with the unique N-terminus of RTN1A. Using a nonbiased approach involving GST pull-downs and MS analysis, we identified the intracellular calcium release channel ryanodine receptor 2 (RyR2) as a direct binding partner of RTN1A. The RyR2 binding site was localized to a highly conserved 150-amino acid residue region. RTN1A displays high preference for RyR2 binding in vitro and in vivo and both proteins colocalize in hippocampal neurons and Purkinje cells. Moreover, we demonstrate the precise subcellular localization of RTN1A in Purkinje cells and show that RTN1A inhibits RyR channels in [3H]ryanodine binding studies on brain synaptosomes. In a functional assay, RTN1A significantly reduced RyR2-mediated Ca2 + oscillations. Thus, RTN1A and RyR2 might act as functional partners in the regulation of cytosolic Ca2 + dynamics the in neurons.
•The reticulon protein RTN1A is an ER-resident protein of unknown function.•We identified the intracellular calcium release channel ryanodine receptor RyR2 as a specific binding partner of RTN1A.•RNT1A co-immunoprecipitates and colocalizes with the ryanodine receptor RyR2 in neurons via its N-terminal domain.•Binding of RTN1A modulates the activity of RyR2.•RTN1A and RyR2 might act as functional partners in the regulation of cytosolic Ca2 + dynamics
RTN, reticulon protein; ER, endoplasmic reticulum; RyR, ryanodine receptor; RHD, reticulon homology domain; CICR, calcium induced calcium release; IPTG, isopropyl-β-d-thiogalactopyranoside; PB, phosphate buffer; DAB, 3,3′-diaminobenzidine tetrahydrochloride dihydrate; CA, cornu ammonis; Reticulon; Brain; Protein–protein interaction; Calcium homeostasis
Uptake of glutamate from the synaptic cleft is mediated by high affinity transporters and is driven by Na+, K+, and H+ concentration gradients across the membrane. Here, we characterize the molecular mechanism of the intracellular pH change associated with glutamate transport by combining current recordings from excitatory amino acid carrier 1 (EAAC1)–expressing HEK293 cells with a rapid kinetic technique with a 100-μs time resolution. Under conditions of steady state transport, the affinity of EAAC1 for glutamate in both the forward and reverse modes is strongly dependent on the pH on the cis-side of the membrane, whereas the currents at saturating glutamate concentrations are hardly affected by the pH. Consistent with this, the kinetics of the pre–steady state currents, measured after saturating glutamate concentration jumps, are not a function of the pH. In addition, we determined the deuterium isotope effect on EAAC1 kinetics, which is in agreement with proton cotransport but not OH− countertransport. The results can be quantitatively explained with an ordered binding model that includes a rapid proton binding step to the empty transporter followed by glutamate binding and translocation of the proton-glutamate-transporter complex. The apparent pK of the extracellular proton binding site is ∼8. This value is shifted to ∼6.5 when the substrate binding site is exposed to the cytoplasm.
glutamate transporter; patch-clamp; laser-pulse photolysis; rapid kinetics; reverse transport
The neuronal glutamate transporter EAAC1 contains several conserved acidic amino acids in its transmembrane domain, which are possibly important in catalyzing transport and/or binding of co/countertransported cations. Here, we have studied the effects of neutralization by site-directed mutagenesis of three of these amino acid side chains, glutamate 373, aspartate 439, and aspartate 454, on the functional properties of the transporter. Transport was analyzed by whole-cell current recording from EAAC1-expressing mammalian cells after applying jumps in voltage, substrate, or cation concentration. Neutralization mutations in positions 373 and 454, although eliminating steady-state glutamate transport, have little effect on the kinetics and thermodynamics of Na+ and glutamate binding, suggesting that these two positions do not constitute the sites of Na+ and glutamate association with EAAC1. In contrast, the D439N mutation resulted in an approximately 10-fold decrease of apparent affinity of the glutamate-bound transporter form for Na+, and an ∼2,000-fold reduction in the rate of Na+ binding, whereas the kinetics and thermodynamics of Na+ binding to the glutamate-free transporter were almost unchanged compared to EAAC1WT. Furthermore, the D439N mutation converted l-glutamate, THA, and PDC, which are activating substrates for the wild-type anion conductance, but not l-aspartate, into transient inhibitors of the EAAC1D439 anion conductance. Activation of the anion conductance by l-glutamate was biphasic, allowing us to directly analyze binding of two of the three cotransported Na+ ions as a function of time and [Na+]. The data can be explained with a model in which the D439N mutation results in a dramatic slowing of Na+ binding and a reduced affinity of the substrate-bound EAAC1 for Na+. We propose that the bound substrate controls the rate and the extent of Na+ interaction with the transporter, depending on the amino acid side chain in position 439.
Electrogenic glutamate transport by the excitatory amino acid carrier 1 (EAAC1) is associated with multiple charge movements across the membrane that take place on time scales ranging from microseconds to milliseconds. The molecular nature of these charge movements is poorly understood at present and, therefore, was studied in this report in detail by using the technique of laser-pulse photolysis of caged glutamate providing a 100-μs time resolution. In the inward transport mode, the deactivation of the transient component of the glutamate-induced coupled transport current exhibits two exponential components. Similar results were obtained when restricting EAAC1 to Na+ translocation steps by removing potassium, thus, demonstrating (1) that substrate translocation of EAAC1 is coupled to inward movement of positive charge and, therefore, electrogenic; and (2) the existence of at least two distinct intermediates in the Na+-binding and glutamate translocation limb of the EAAC1 transport cycle. Together with the determination of the sodium ion concentration and voltage dependence of the two-exponential charge movement and of the steady-state EAAC1 properties, we developed a kinetic model that is based on sequential binding of Na+ and glutamate to their extracellular binding sites on EAAC1 explaining our results. In this model, at least one Na+ ion and thereafter glutamate rapidly bind to the transporter initiating a slower, electroneutral structural change that makes EAAC1 competent for further, voltage-dependent binding of additional sodium ion(s). Once the fully loaded EAAC1 complex is formed, it can undergo a much slower, electrogenic translocation reaction to expose the substrate and ion binding sites to the cytoplasm.
glutamate transporter; charge movement; patch clamp; caged compounds; rapid kinetics
The cervical facet joint and its capsule are a common source of neck pain from whiplash. Mechanical hyperalgesia elicited by painful facet joint distraction is associated with spinal neuronal hyperexcitability that can be induced by transmitter/receptor systems that potentiate the synaptic activation of neurons. This study investigated the temporal response of a glutamate receptor and transporters in the dorsal root ganglia (DRG) and spinal cord. Bilateral C6/C7 facet joint distractions were imposed in the rat either to produce behavioral sensitivity or without inducing any sensitivity. Neuronal metabotropic glutamate receptor-5 (mGluR5) and protein kinase C-epsilon (PKCε) expression in the DRG and spinal cord were evaluated on days 1 and 7. Spinal expression of a glutamate transporter, excitatory amino acid carrier 1 (EAAC1), was also quantified at both time points. Painful distraction produced immediate behavioral hypersensitivity that was sustained for 7 days. Increased expression of mGluR5 and PKCε in the DRG was not evident until day 7 and only following painful distraction; this increase was observed in small-diameter neurons. Only painful facet joint distraction produced a significant increase (p<0.001) in neuronal mGluR5 over time, and this increase also was significantly elevated (p ≤ 0.05) over responses in the other groups at day 7. However, there were no differences in spinal PKCε expression on either day or between groups. Spinal EAAC1 expression was significantly increased (p<0.03) only in the nonpainful groups on day 7. Results from this study suggest spinal glutamatergic plasticity is selectively modulated in association with facet-mediated pain.
mechanical hyperalgesia; glutamate receptor; glutamate transporter; protein kinase C; facet; spinal cord
To define the changes in gene and protein expression of the neuronal glutamate transporter (EAAT3/EAAC1) in a rat model of temporal lobe epilepsy as well as in human hippocampal and neocortical epilepsy.
The expression of EAAT3/EAAC1 mRNA was measured by reverse Northern blotting in single dissociated hippocampal dentate granule cells from rats with pilocarpine-induced temporal lobe epilepsy (TLE) and age-matched controls, in dentate granule cells from hippocampal surgical specimens from patients with TLE, and in dysplastic neurons microdissected from human focal cortical dysplasia specimens. Immunolabeling of rat and human hippocampi and cortical dysplasia tissue with EAAT3/EAAC1 antibodies served to corroborate the mRNA expression analysis.
The expression of EAAT3/EAAC1 mRNA was increased by nearly threefold in dentate granule cells from rats with spontaneous seizures compared with dentate granule cells from control rats. EAAT3/EAAC1 mRNA levels also were high in human dentate granule cells from patients with TLE and were significantly elevated in dysplastic neurons in cortical dysplasia compared with nondysplastic neurons from postmortem control tissue. No difference in expression of another glutamate transporter, EAAT2/GLT-1, was observed. Immunolabeling demonstrated that EAAT3/EAAC1 protein expression was enhanced in dentate granule cells from both rats and humans with TLE as well as in dysplastic neurons from human cortical dysplasia tissue.
Elevations of EAAT3/EAAC1 mRNA and protein levels are present in neurons from hippocampus and neocortex in both rats and humans with epilepsy. Upregulation of EAAT3/EAAC1 in hippocampal and neocortical epilepsy may be an important modulator of extracellular glutamate concentrations and may occur as a response to recurrent seizures in these cell types.
Glutamate transporter; EAAT3/EAAC1; Epilepsy; Dentate; Dysplasia
In the mammalian brain, the specificity of excitatory synaptic transmission depends on rapid diffusion of glutamate away from active synapses and the powerful uptake capacity of glutamate transporters in astrocytes. The extent to which neuronal glutamate transporters influence the lifetime of glutamate in the extracellular space (ECS) remains unclear. Here we show that EAAC1, the predominant neuronal glutamate transporter at excitatory synapses in hippocampal area CA1, buffers glutamate released during synaptic events and prolongs the time course of its clearance by astrocytes. EAAC1 does not significantly alter activation of receptors in the synaptic cleft. Instead, it reduces recruitment of peri/extrasynaptic NR2-Bcontaining NMDA receptors (NR2B-NMDARs), thereby facilitating induction of long-term potentiation (LTP) by short bursts of high-frequency stimulation. We describe novel roles of EAAC1 in regulating glutamate diffusion and propose that NMDARs at different subsynaptic locations can make distinct contributions to the regulation of synaptic strength.
Glutamate; Glutamate receptor; Glutamate transporters; Hippocampus; Diffusion; synaptic transmission; Synaptic plasticity
Solute carrier family 1, member 1 (SLC1A1; also known as EAAT3 and EAAC1) is the major epithelial transporter of glutamate and aspartate in the kidneys and intestines of rodents. Within the brain, SLC1A1 serves as the predominant neuronal glutamate transporter and buffers the synaptic release of the excitatory neurotransmitter glutamate within the interneuronal synaptic cleft. Recent studies have also revealed that polymorphisms in SLC1A1 are associated with obsessive-compulsive disorder (OCD) in early-onset patient cohorts. Here we report that SLC1A1 mutations leading to substitution of arginine to tryptophan at position 445 (R445W) and deletion of isoleucine at position 395 (I395del) cause human dicarboxylic aminoaciduria, an autosomal recessive disorder of urinary glutamate and aspartate transport that can be associated with mental retardation. These mutations of conserved residues impeded or abrogated glutamate and cysteine transport by SLC1A1 and led to near-absent surface expression in a canine kidney cell line. These findings provide evidence that SLC1A1 is the major renal transporter of glutamate and aspartate in humans and implicate SLC1A1 in the pathogenesis of some neurological disorders.
In the central nervous system, electrogenic sodium- and potassium-coupled glutamate transporters terminate the synaptic actions of this neurotransmitter. In contrast to acidic amino acids, dicarboxylic acids are not recognized by glutamate transporters, but the related bacterial DctA transporters are capable of transporting succinate and other dicarboxylic acids. Transmembrane domain 8 contains several residues that differ between these two types of transporters. One of these, aspartate-444 of the neuronal glutamate transporter EAAC1, is conserved in glutamate transporters, but a serine residue occupies this position in DctA transporters. When aspartate-444 is mutated to serine, cysteine, alanine, or even to glutamate, uptake of d-[3H]-aspartate as well as the inwardly rectifying steady-state currents induced by acidic amino acids is impaired. Even though succinate was not capable of inducing any steady-state transport currents, the dicarboxylic acid inhibited the sodium-dependent transient currents by the mutants with a neutral substitution at position 444. In the neutral substitution mutants inhibition of the transients was also observed with acidic amino acids. In the D444E mutant, acidic amino acids were potent inhibitors of the transient currents, whereas the apparent affinity for succinate was lower by at least three orders of magnitude. Even though L-aspartate could bind to D444E with a high apparent affinity, this binding resulted in inhibition rather than stimulation of the uncoupled anion conductance. Thus, a carboxylic acid–containing side chain at position 444 prevents the interaction of glutamate transporters with succinate, and the presence of aspartate itself at this position is crucial for productive substrate binding compatible with substrate translocation.
In the central nervous system, excitatory amino acid transporters (EAATs) localized to neurons and glia terminate the actions of synaptically released glutamate. Whereas glial transporters are primarily responsible for maintaining low ambient levels of extracellular glutamate, neuronal transporters have additional roles in shaping excitatory synaptic transmission. Here we test the hypothesis that the expression level of the Purkinje cell (PC)-specific transporter, EAAT4, near parallel fiber (PF) release sites controls the extrasynaptic glutamate concentration transient following synaptic stimulation. Expression of EAAT4 follows a parasagittal banding pattern that allows us to compare regions of high and low EAAT4-expressing PCs. Using EAAT4 promoter driven eGFP reporter mice together with pharmacology and genetic deletion, we show that the level of neuronal transporter expression influences extrasynaptic transmission from PFs to adjacent Bergmann glia (BG). Surprisingly, a twofold difference in functional EAAT4 levels is sufficient to alter signaling to BG although EAAT4 may only be responsible for removing a fraction of released glutamate. These results demonstrate that physiological regulation of neuronal transporter expression can alter extrasynaptic neuro-glial signaling.
synaptic transmission; Purkinje cell; parallel fiber; EAAT4
The reticulon family is a diverse group of proteins that mostly localize to the endoplasmic reticulum and may be important in neurodegenerative diseases.
The reticulon family is a large and diverse group of membrane-associated proteins found throughout the eukaryotic kingdom. All of its members contain a carboxy-terminal reticulon homology domain that consists of two hydrophobic regions flanking a hydrophilic loop of 60-70 amino acids, but reticulon amino-terminal domains display little or no similarity to each other. Reticulons principally localize to the endoplasmic reticulum, and there is evidence that they influence endoplasmic reticulum-Golgi trafficking, vesicle formation and membrane morphogenesis. However, mammalian reticulons have also been found on the cell surface and mammalian reticulon 4 expressed on the surface of oligodendrocytes is an inhibitor of axon growth both in culture and in vivo. There is also growing evidence that reticulons may be important in neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis. The diversity of structure, topology, localization and expression patterns of reticulons is reflected in their multiple, diverse functions in the cell.
The cervical facet joint and its capsule have been reported to be injured during whiplash scenarios and are a common source of chronic neck pain from whiplash. Both the metabotropic glutamate receptor 5 (mGluR5) and the excitatory amino acid carrier 1 (EAAC1) have pivotal roles in chronic pain. In this study, spinal mGluR5 and EAAC1 were quantified following painful facet joint distraction in a rat model of facet-mediated painful loading and were evaluated for their correlation with the severity of capsule loading. Rats underwent either a dynamic C6/C7 joint distraction simulating loading experienced during whiplash (distraction; n = 12) or no distraction (sham; n = 6) to serve as control. The severity of capsular loading was quantified using strain metrics, and mechanical allodynia was assessed after surgery. Spinal cord tissue was harvested at day 7 and the expression of mGluR5 and EAAC1 were quantified using Western blot analysis. Mechanical allodynia following distraction was significantly (p < 0.001) higher than sham. Spinal expression of mGluR5 was also significantly (p < 0.05) greater following distraction relative to sham. However, spinal EAAC1 was significantly (p = 0.0003) reduced compared to sham. Further, spinal mGluR5 expression was significantly positively correlated to capsule strain (p < 0.02) and mechanical allodynia (p < 0.02). Spinal EAAC1 expression was significantly negatively related to one of the strain metrics (p < 0.003) and mechanical allodynia at day 7 (p = 0.03). These results suggest that the spinal glutamatergic system may potentiate the persistent behavioral hypersensitivity that is produced following dynamic whiplash-like joint loading; chronic whiplash pain may be alleviated by blocking mGluR5 expression and/or enhancing glutamate transport through the neuronal transporter EAAC1.
excitatory amino acid carrier 1; facet; glutamate; metabotropic glutamate receptor 5; pain
The SLC1A1 gene, which encodes the neuronal glutamate transporter, EAAC1, has consistently been implicated in obsessive-compulsive disorder (OCD) in genetic studies. Moreover, neuroimaging, biochemical and clinical studies support a role for glutamatergic dysfunction in OCD. Although SLC1A1 is an excellent candidate gene for OCD, little is known about its regulation at the genomic level. Here, we report the identification and characterization of three alternative SLC1A1/EAAC1 mRNAs: a transcript derived from an internal promoter, termed P2 to distinguish it from the transcript generated by the primary promoter (P1), and two alternatively spliced mRNAs: ex2skip, which is missing exon 2, and ex11skip, which is missing exon 11. All isoforms inhibit glutamate uptake from the full-length EAAC1 transporter. Ex2skip and ex11skip also display partial colocalization and interact with the full-length EAAC1 protein. The three isoforms are evolutionarily conserved between human and mouse, and are expressed in brain, kidney and lymphocytes under nonpathological conditions, suggesting that the isoforms are physiological regulators of EAAC1. Moreover, under specific conditions, all SLC1A1 transcripts were differentially expressed in lymphocytes derived from subjects with OCD compared with controls. These initial results reveal the complexity of SLC1A1 regulation and the potential clinical utility of profiling glutamatergic gene expression in OCD and other psychiatric disorders.
EAAC1; EAAT3; internal promoter; obsessive-compulsive disorder; protein isoform; SLC1A1
In the brain, glutamate is an extracellular transmitter that mediates cell-to-cell communication. Prior to synaptic release it is pumped into vesicles by vesicular glutamate transporters (VGLUTs). To inactivate glutamate receptor responses after release, glutamate is taken up into glial cells or neurons by excitatory amino acid transporters (EAATs). In the pancreatic islets of Langerhans, glutamate is proposed to act as an intracellular messenger, regulating insulin secretion from β-cells, but the mechanisms involved are unknown. By immunogold cytochemistry we show that insulin containing secretory granules express VGLUT3. Despite the fact that they have a VGLUT, the levels of glutamate in these granules are low, indicating the presence of a protein that can transport glutamate out of the granules. Surprisingly, in β-cells the glutamate transporter EAAT2 is located, not in the plasma membrane as it is in brain cells, but exclusively in insulin-containing secretory granules, together with VGLUT3. In EAAT2 knock out mice, the content of glutamate in secretory granules is higher than in wild type mice. These data imply a glutamate cycle in which glutamate is carried into the granules by VGLUT3 and carried out by EAAT2. Perturbing this cycle by knocking down EAAT2 expression with a small interfering RNA, or by over-expressing EAAT2 or a VGLUT in insulin granules, significantly reduced the rate of granule exocytosis. Simulations of granule energetics suggest that VGLUT3 and EAAT2 may regulate the pH and membrane potential of the granules and thereby regulate insulin secretion. These data suggest that insulin secretion from β-cells is modulated by the flux of glutamate through the secretory granules.